Synthesis of mRNAs in eukaryotic cells is a highly complex process, including, in addition to transcription itself, splicing and 3' end formation of mRNA precursors. My laboratory has studied the mechanisms and regulation of these processes for many years, and also shown how they are integrated with other cellular events such as DNA damage, and also how they function in differentiation and disease. This proposal combines our studies in these areas, and can be divided into the following three broad areas: 1) mRNA processing and disease. Studies examining the roles of several proteins implicated in RNA metabolism in cancer and neurodegenerative disease will be pursued. The role of RNA processing factors in cancer reflects two distinct mechanisms, overexpression of splicing regulatory proteins and mutation of genes encoding various splicing factors. With respect to the former, ongoing experiments stemming from studies elucidating the mechanism underlying deregulation of PKM alternative splicing (AS) in cancer will be pursued. Important questions include the generality of the mechanism, the importance of splicing factor overexpression to tumor development, and the identity of the critical regulated splicing events. Splicing factor mutations can cause MDS and certain leukemias, and ongoing studies are aimed at elucidating mechanisms by which mutations affect the function of several, e.g., SRSF2 and SF3B1, and how such defects lead to disease. With respect to neurodegenerative disease, work centers on how ALS-causing mutations in the RNA/DNA-binding protein TLS/FUS disrupt protein function; how the hexanucleotide repeat expansions in C9ORF72 form G-quadruplex structures that sequester hnRNP H, disrupt AS, and contribute to ALS; and how disease-causing mutations in the RNA/DNA helicase Senataxin affect its role in prevention of transcription-induced DNA damage. 2) mRNA processing and differentiation. Studies examining how changes in AS and alternative polyadenylation (APA) contribute to human embryonic stem cell pluripotency and differentiation will be pursued. How AS affects the function of the transcriptional regulator TCF3 and how this contributes to differentiation will be determined. The mechanism of the AS event, including how the splicing regulators are themselves regulated, will be investigated. Our unexpected finding that AS of transcripts encoding two PA factors, both subunits of CPSF and implicated in AAUAAA recognition, is altered during differentiation will be pursued. How the distinct isoforms affect APA, and differentiation, will be determined, as will the mechanism by which they influence PA site choice. 3) RNA processing and transcription. Experiments examining the function of the RNA pol II CTD, a unique domain consisting of 26-52 heptad repeats (consensus YSPTSPS), will be continued. For example, recent experiments establishing a role for P-Tyr1 residues in facilitating turnover of certain lncRNAs, and suggesting that this involves interaction with the RNA helicase Mtr4 and the PA machinery, will be pursued. These and other experiments will advance our understanding of the CTD code that links transcription and RNA processing.